Artefact reduction for angularly-selective illumination

ABSTRACT

An optical device includes a sample holder configured to fix an object in the beam path of the optical device, and an illumination module which has a plurality of light sources and configured to illuminate the object from a plurality of illumination directions by operating the light sources, wherein each illumination direction has an assigned luminous field. The optical device also has a filter arranged between the illumination module and the sample holder and configured to expand the assigned luminous field for each illumination direction. As a result, it is possible to reduce artefacts on account of contaminants during the angularly-selective illumination. Techniques of digital artefact reduction are also described. By way of example, the optical device can be a microscope.

RELATED APPLICATIONS

The present application is a U.S. National Stage application ofInternational PCT Application No. PCT/EP2017/059949 filed on Apr. 26,2017 which claims priority benefit of German Application No. DE 10 2016108 079.9 filed on May 2, 2016, the contents of each are incorporated byreference in their entirety.

FIELD OF THE INVENTION

Various embodiments of the invention relate to an optical device havinga filter, which is arranged between an illumination module of theoptical device which has a plurality of light sources and a sampleholder of the optical device and which is configured to expand theassigned luminous field for each illumination direction. Various furtherembodiments of the invention relate to a method in which an artefactreduction is carried out for each of a plurality of measurement imagesbefore the various measurement images are subsequently combined in orderto obtain a result image. By way of example, the result image can have aphase contrast.

BACKGROUND

In the optical imaging of objects it may often be worthwhile to generatea so-called phase contrast image. In a phase contrast image, at leastpart of the image contrast is caused by a phase shift of the lightthrough the imaged object. It is thus possible to image withcomparatively higher contrast in particular such objects which bringabout no or only a small attenuation of the amplitude of the light, buta significant phase shift (phase objects). Biological samples as objectin a microscope may typically bring about a larger change in phase thanchange in amplitude of the electromagnetic field.

Various techniques for phase contrast imaging are known, for instancedark-field illumination, oblique illumination, differential interferencecontrast (DIC) or Zernike phase contrast. Further techniques would bee.g. the so-called knife edge method or helical phase contrast.

Such techniques mentioned above have various disadvantages orlimitations. Thus, the DIC technique, the Zernike technique, the knifeedge method and helical phase contrast typically necessitate providing,by comparison with conventional amplitude imaging, additional opticalelements between sample and detector in the region of the so-calleddetection optics. This can lead to structural limitations particularlyin the case of modularly constructed microscopes. Costs are typicallyincreased. In the case of thin samples, typically only a few photonscontribute to image generation in the case of dark-field illumination,which can lead to noisy images of lower quality. A subsequent evaluationor analysis of the images may not be possible, or may be possible onlyto a limited extent. Oblique illumination typically leads to anasymmetrical increase in contrast, which can in turn bring about areduced quality of the images.

Therefore, techniques for generating a phase contrast image by digitalpost-processing are known as well. By way of example, DE 10 2014 112 242A1 discloses techniques for generating a phase contrast image bycombining a plurality of captured intensity images. In this case, thevarious intensity images are associated with different illuminationdirections. Such techniques are sometimes referred to asangularly-selective illumination.

In the case of phase contrast imaging by means of angularly-selectiveillumination it can happen that contaminants in the beam path adverselyaffect the quality of the phase contrast image. In particular, it hasbeen observed that contaminants in the beam path can be manifested as anextensive pattern in the phase contrast image if they are arranged at adistance from a focal plane of an objective of the optical device (indefocused fashion).

Corresponding disadvantages can also occur for other imaging techniquesin association with angularly-selective illumination, e.g. inassociation with bright-field imaging.

SUMMARY

Therefore, there is a need for improved techniques for imaging by meansof angularly-selective illumination. Angularly-selective illumination issometimes also referred to as structured illumination. In particular,there is a need for such techniques which make it possible to reduceartefacts on account of contaminants in the beam path.

This object is achieved by the features of the independent patentclaims. The features of the dependent patent claims define embodiments.

In one example, an optical device includes a sample holder, anillumination module and a filter. The sample holder is configured to fixan object in the beam path of the optical device. The illuminationmodule has a plurality of light sources. The illumination module isconfigured to illuminate the object from a plurality of illuminationdirections by operating the light sources. Each illumination directionhas an assigned luminous field. The filter is arranged between theillumination module and the sample holder. The filter is configured toexpand the assigned luminous field for each illumination direction.

By way of example, the optical device can be a microscope. It would bepossible, for example, for the optical device to be configured forreflected-light imaging and/or for transmitted-light imaging. Theoptical device can have, for example, an eyepiece and/or an objective.The optical device could include, for example, a further illuminationmodule, for example having a laser for fluorescence imaging.

The object can be for example a phase object, such as a biologicalsample, for example. By way of example, a corresponding biologicalsample could have one or more cell cultures.

The luminous field can describe for example the quantity of light foreach associated illumination direction which is present at differentpoints in the space. The luminous field can be associated e.g. with apropagation direction of the light. The luminous field can have forexample a specific defined width between two edges; by way of example,the width of the luminous field can be defined perpendicular to thepropagation direction. The luminous field can define e.g. the beam path.The beam path can have e.g. a central axis that is defined symmetricallywith respect to the edges of the luminous field.

By expanding the luminous field, the width thereof can be increased.This means that the extent of the luminous field has the effect thatlight is present with a greater solid angle. What can thereby beachieved, for example, is that the object is illuminated from anextended solid angle which is centered for example around the respectiveillumination direction. As a result of the expanding, the luminous fieldof the different illumination directions can therefore have acomparatively large extent perpendicular to the light propagationdirection in the region of the sample holder or of the object.

What can be achieved by such techniques is that artefacts in a resultimage on account of contaminants in the beam path are reduced or areremoved (artefact reduction). In particular, artefacts in the resultimage on account of contaminants which are situated outside a focalplane of the optical device in the region of the sample holder can bereduced. In particular, it may be possible to reduce artefacts onaccount of contaminants situated outside the focal plane, while at thesame time the quality of the imaging with respect to the object, whichis arranged in the region of the focal plane of the optical device, isnot, or not significantly, reduced. In this case, the quality of theimaging can be characterized for example by an edge sharpness of theobject, a signal-to-noise ratio, image noise, etc.

Various filters can be used to achieve the expansion of the luminousfield for the different illumination directions. By way of example, thefilter could have a diffusing plate or be implemented as a diffusingplate. A particularly robust filter that is simple to produce can beused in this way.

By way of example, the diffusing plate could be implemented by a plasticplate. By way of example, it would be possible for one or more surfacesof the diffusing plate to have a roughened structure, i.e. for a surfacehaving a significant topology to be present. In this case, a lengthscale of the topology of the surface can correlate with a length scaleof the expansion of the luminous field. The filter can bring about theexpansion of the luminous field for both transmission directions or elseonly for light which is incident on the filter from the illuminationmodule.

It may be worthwhile for the filter to have a comparatively hightransmittance for the light incident along the respective illuminationdirection. For example, the transmittance can be >50%, preferably >85%,particularly preferably >95%. What can be achieved thereby is that theprovision of the filter does not cause the quality of the imaging to bereduced or significantly reduced.

For example, it would be possible for a surface of the diffusing plateto form a significant angle with a central axis of the beam paths whichare associated with the different illumination directions. For example,it would be possible for said angle to be 50°, preferably more than 70°,particularly preferably more than 85°. Such a perpendicular arrangementmakes it possible to reduce the reflection on the surface of the filterand to increase the transmittance.

For example, it would be possible for the diffusing plate to bepositioned near the illumination module. For example, a distance betweena surface of the diffusing plate and the illumination module could besmaller than an extent of the surface of the diffusing plate. Forexample, a distance between a surface of the diffusing plate and theillumination module could be smaller than the width of the luminousfield for different illumination directions upstream or optionallydownstream of the filter.

In one example, it would be possible for the illumination module to havea carrier, on which the light sources are fitted. It would then bepossible for the filter to be rigidly coupled to the carrier. In onesuch example, it may be possible to position the filter in particularlyclose local relationship with respect to the illumination module. Aparticularly efficient expansion of the luminous field of the differentillumination directions can be achieved as a result. Furthermore, thefilter can be arranged efficiently in respect of structural space.

The optical device can furthermore have the detector, e.g. acomplimentary metal-oxide-semiconductor, used as an image sensor andcommonly referred to as a “CMOS” detector or a charge coupled device(“CCD”) detector or a photomultiplier. The detector can have an array ofpixels. The detector can be arranged in the beam path of the opticaldevice. The optical device can also have a computing unit. The computingunit can be configured to control the illumination module forilluminating the object from a plurality of measurement illuminationdirections. The computing unit can furthermore be configured to controlthe detector for capturing measurement images of the object. In thiscase, the measurement images are assigned to the measurementillumination directions. The computing unit can furthermore beconfigured to combine the measurement images in order to obtain a resultimage.

Different assignments between the measurement illumination directionsand the measurement images are possible in the various examplesdescribed herein. By way of example, a 1:1 assignment would be possible.By way of example, the computing unit could be configured to control thedetector for capturing a respective measurement image of the object foreach of the measurement illumination directions. However, a differentassignment would also be possible: by way of example, the computing unitcould be configured to control the detector such that a measurementimage is assigned to more than one measurement illumination direction.In the case of a 1:n assignment where n>2, the imaging speed can beincreased: that may be worthwhile particularly in the case ofbright-field imaging.

By way of example, it would be possible for the result image to have aphase contrast. However, it would also be possible for the result imageto have no, or no significant, phase contrast. By way of example,traditional bright-field imaging can be operated in this case.

In the case of bright-field imaging, it would be possible, for example,for the computing unit to be configured to control the illuminationmodule for illuminating the object from a plurality of measurementillumination directions in a time-parallel manner. The computing unitcan then furthermore be configured to control the detector for capturinga result image. The result image can have a bright-field contrast sincean illumination is carried out simultaneously from differentillumination directions. In this case, it may be worthwhile, forexample, to illuminate the object as uniformly as possible from thedifferent spatial directions. This may necessitate activating a largenumber or all of the available light sources in a time-parallel manner.

The different measurement illumination directions can correspond to theselective activation of different light sources of the illuminationmodule. By way of example, the object can be illuminated sequentiallyfrom the different measurement illumination directions. Alternatively oradditionally it would be possible for the different measurementillumination directions to be assigned to different colors and/orpolarizations, such that it is possible in this way to effect aseparation between the different illumination directions for themeasurement images. During sequential illumination, per sequence step ineach case one or more illumination directions can be implemented byoperating one or more light sources, i.e. a 1:n assignment where n>=1can be implemented. In this case, n can vary or be identical fordifferent measurement images.

In this case, it may be possible, for example, for illuminationdirections that are different as much as possible to be used for thedifferent measurement images, i.e. illumination directions that form alarge angle with one another. What can be achieved thereby is that theresult image has particularly strong phase contrast with respect to theobject. In this case, it is possible to apply for example techniquessuch as are known in principle from DE 10 2014 112 242 A1; thecorresponding disclosure content is incorporated by cross-referenceherein.

In accordance with such examples described above which are based on theuse of a filter that expands the corresponding luminous field for eachillumination direction, a particularly rapid, hardware-implementedreduction of artefacts associated with contaminants can be carried out.In particular, with regard to the artefact reduction it may beunnecessary to carry out an additional digital post-processing of themeasurement images and/or of the result image. The hardwareimplementation of the artefact reduction makes it possible to prevent anadditional latency from being introduced into the digitalpost-processing on account of further processing steps. As a result, itmay be possible to provide result images particularly rapidly; inparticular, it may be possible to implement real-time applications ofthe phase contrast imaging.

Such examples described above concerning the use of a filter forhardware-based artefact reduction can be replaced by or combined withtechniques of digital artefact reduction. In the case of digitalartefact reduction, it may be possible, in the context of the digitalpost-processing, to reduce artefacts on account of contaminants arrangedin defocused fashion in the beam path of the optical device. The variousexamples concerning the digital artefact reduction and thehardware-implemented artefact reduction can be combined with oneanother.

In one example, a method includes driving an illumination module of anoptical device for illuminating, for example sequentially, an objectfrom a plurality of measurement illumination directions. Theillumination module has a plurality of light sources. The methodfurthermore includes: driving a detector of the optical device forcapturing measurement images of the object, wherein the measurementimages are assigned to the measurement illumination directions. Themethod also includes, for each measurement image: carrying out anartefact reduction which reduces an artefact in the respectivemeasurement image on account of a contaminant arranged in defocusedfashion. The method further includes, after carrying out the artefactreduction for each measurement image: combining the measurement imagesin order to obtain a result image.

By way of example, it would be possible for the result image to have aphase contrast. However, it would also be possible for the result imageto have no, or no significant, phase contrast. By way of example,traditional bright-field imaging can be operated in this case.

By way of example, an assigned measurement image could be captured foreach measurement illumination direction. It would also be possible formore than one measurement illumination direction to be activated for atleast some measurement images. A 1:n assignment where n>=1 can thus beimplemented. In this case, n can vary or be identical for differentmeasurement images.

As an alternative or in addition to a sequential illumination of theobject from the plurality of measurement illumination directions, itwould also be possible to achieve a separation of the illuminationdirections for the measurement images by way of the color (or spectralrange) and/or the polarization of the light which is associated with thedifferent measurement illumination directions.

With regard to combining the measurement images in order to obtain theresult image, once again it is possible to apply techniques such as areknown in principle from DE 10 2014 112 242 A1. As a result, it ispossible to generate a phase contrast for the result image. For example,a number of two, four, eight or more measurement images can be capturedand combined with one another in order to obtain the result image. Inthis case, by way of example, weighted sums can be used. In this case,corresponding weighting factors can assume positive and/or negativevalues.

What can be achieved by carrying out the artefact reduction with respectto each measurement image is that the result image has no, or nosignificant, artefacts on account of contaminants. In particular, theearly artefact reduction with respect to each measurement image makes itpossible to prevent a situation in which, on account of the combinationof the different measurement images in order to obtain the result image,the artefacts from the different measurement images are transferred tothe result image.

In this case, various techniques can be used for carrying out theartefact reduction. In one example, it would be possible for theartefact reduction to be carried out solely on the basis of informationobtained from the different measurement images; this may mean that it isnot necessary to capture additional reference images of the object inorder to carry out the artefact reduction. By way of example, asoft-focus filter could be employed in regions outside the object inorder to reduce the artefacts. This may afford the advantage of areduced exposure of the object to light, which may be advantageous forexample with regard to biological samples. Moreover, the time durationrequired for carrying out the measurement until obtaining the resultimage (measurement duration) can be reduced as a result.

In other examples, however, it would also be possible to take account ofadditional information—over and above the measurement images—forcarrying out the artefact reduction. For example, it would be possiblefor the method to further include for each measurement image: drivingthe illumination module for illuminating—for example sequentially—theobject from at least one assigned reference illumination direction. Themethod can then include, for each reference illumination direction:driving the detector for capturing a reference image of the object. Themethod can then also include, when carrying out the artefact reduction,for each measurement image: combining the respective measurement imagewith the at least one assigned reference image in order to obtain atleast one correction image which is indicative of the artefact.

In other words, it may thus be possible that, for each measurementimage, in each case one or more assigned reference images are capturedfor corresponding reference illumination directions. On the basis ofsaid reference images, it may be possible to obtain for each measurementimage one or more correction images indicating the artefact in therespective measurement image; on the basis of the correction images, itmay then be possible to identify the artefact in the respectivemeasurement image and, on the basis thereof, to correct the respectivemeasurement image in order to remove the artefact or to reduce aninfluence of the artefact on the measurement image.

Such techniques may in particular exploit the fact that as a result ofthe defocused arrangement of the contaminant, it is possible to obtain acharacteristic position change of the artefact in the differentreference images—in particular in comparison with the position change ofthe object in the different reference images. Through a suitable choiceof the reference illumination directions, it may be possible toconfigure this position change of the artefact in a particularlycharacteristic manner, which may enable a particularly accurateidentification of the artefact in the measurement image.

For example, what may be achieved is that between the at least onereference image and the associated measurement image the artefact has alarger position change than the object itself. In particular, through asuitable choice of the reference illumination directions, it may bepossible that the object has no, or no significant, position changebetween the at least one reference image and the associated measurementimage; while the artefact has a significant position change between theat least one reference image and the associated measurement image.

On account of the characteristic position change, by combining therespective measurement image with the at least one assigned referenceimage it is possible to obtain the correction image in such a way thatthe correction image is indicative of the artefact. In particular, itmay be possible for the artefacts to have a greater intensity than therespective objects in the different correction images. Therefore, on thebasis of an intensity threshold value, the artefacts can be separatedfrom other image constituents, such as, for example, the object or thebackground.

By using the reference images, it is possible to obtain additionalinformation about the artefact. In particular, it may be possible toisolate the artefact from other constituents of the measurement imagefor example the object and the background. As a result, the artefactreduction can be carried out particularly accurately, without otherimage constituents being adversely influenced.

For example, it would be possible for the method to include, whencarrying out the artefact reduction, for each correction image: applyingan image segmentation on the basis of an intensity threshold value inorder to obtain an isolated artefact region in the correction image. Theartefact region can include the artefact. It may then be possible, whencarrying out the artefact reduction, for each measurement image, toremove the artefact on the basis of the artefact region of therespective correction image.

By means of the image segmentation, it is possible to decompose therespective correction image into two or more continuous regions. In thiscase, the different continuous regions can be defined with respect tothe intensity threshold value. The intensity threshold value can takeaccount of positive and/or negative amplitudes—for instance a comparisonwith a center point of the intensity values of different pixels of therespective correction image. With respect to the intensity thresholdvalue it is also possible to take account of tolerances, for example inorder to ensure transitions between the different continuous regions inaccordance with specific boundary conditions. As a result of the imagesegmentation it is possible to obtain the artefact region, which makesit possible to mark the artefact in the correction image. By virtue ofsuch a marking, it may then be possible particularly easily to reducethe artefact in the measurement image.

In various examples, it is possible for in each case a single referenceimage to be captured per measurement image. It may then be possible tocarry out a particularly rapid artefact reduction without significantlylengthening the measurement duration. Real-time applications arepossible, for example. At the same time, however, it may be possible forthe accuracy of the artefact reduction to be limited by the limitednumber of reference images. Therefore, in other examples it may bepossible for more than a single reference image to be captured permeasurement image, for example a number of two, three or four referenceimages.

In one example, the contaminants can have scatterers and absorbers.Typically, both scatterers and absorbers have a high intensity in thecaptured images; in this case, however, a sign of the amplitude isdifferent for scatterers and absorbers, e.g. with respect to a meanvalue of the amplitudes of the different pixels and/or with respect to avalue of the amplitudes of pixels which image the background. This meansthat it is possible, for example, for scatterers to appear with brightcontrast in the image; while absorbers appear with dark contrast in theimage. In such an example, in particular, it may be possible for morethan a single reference image to be captured per measurement image inorder to carry out as accurate an artefact reduction as possible bothfor the artefacts on account of scatterers and for the artefacts onaccount of absorbers.

For example, it would then be possible to carry out, for each correctionimage, a correction of the respective artefact region on the basis ofthe artefact region of a further correction image. In this case, it ispossible to carry out the correction of the artefact region for suchpairs of correction images which are associated with the samemeasurement image.

For example, the correction of the different artefact regions couldserve for separating artefact regions which are assigned either toscatterers or to absorbers. As a result, it may be possible to correctthe measurement image particularly accurately; in particular, it ispossible to avoid a mixing of artefacts on account of scatterers orabsorbers.

A description has been given above of techniques in which a correctionimage that is indicative of the artefact is obtained on the basis of acombination of the respective measurement image with the at least oneassigned reference image. In addition, or as an alternative to suchtechniques, other implementations of the artefact reduction are alsopossible.

In one example, the method includes, for each measurement image: drivingthe illumination module for illuminating, for example sequentially, theobject from an assigned sequence of reference illumination directions.For each reference illumination direction, the method further includes:driving the detector for capturing a reference image of the object. Whencarrying out the artefact reduction, the method also includes, for eachmeasurement image: identifying a movement of the artefact as a functionof the respective sequence of the reference illumination directions inthe assigned reference images. The respective artefact reduction isbased on the respectively identified movement of the artefact.

The sequence of reference illumination directions can also be processedat least partly in a time-parallel manner, e.g. by superimposition oflight having different colors and/or different polarizations. Thedifferent reference images can be separated in this way.

In this case, it is possible to configure the movement of the artefactas a function of the respective sequence in a characteristic manner. Inparticular, it is possible for the movement of the artefact to include asequence of incremental position changes for the sequence of thereference illumination directions, wherein the movement of the artefactthat is formed in this way is different than the corresponding movementof the object as a function of the respective sequence. In particular,it may be possible that, through a suitable choice of the referenceillumination directions, a particularly small or no movement of theobject is obtained as a function of the sequence. The movement of theartefact can be adapted by a suitable choice of the sequence.

By way of example, it would be possible to identify the movement of theobject and/or the movement of the artefact as a function of therespective sequence of the reference illumination directions on thebasis of image segmentation and/or edge recognition techniques. By wayof example, alternatively or additionally it would be possible toidentify the movement of the object and/or the movement of the artefacton the basis of prior knowledge. For example, the prior knowledgedepending on the reference illumination directions can describe, forexample qualitatively or quantitatively, the expected movement of theobject and/or of the artefact. By way of example, it would be possibleto identify the movement of the object and/or the movement of theartefact on the basis of an optimization. For example, for instance incombination with the abovementioned techniques of image segmentation,edge recognition and/or prior knowledge, an iterative optimization thatdetermines the movement of the object and/or the movement of theartefact could be carried out. The optimization can be associated with atermination criterion, for example, which concerns for example therequired time duration, the number of iterations and/or an accuracy ofthe correspondence to the prior knowledge.

By way of example, XUE, T., RUBINSTEIN M., LIO C., FREEMAN W. T. “Acomputational approach for obstruction-free photography” in ACM Trans.Graph. Proc. ACM SIGGRAPH 2015, 34 (2015) 79, discloses techniques forseparating a reflective foreground or a concealing foreground of animage from a background of the image. This makes use of a movement ofthe camera and a resultant movement of the foreground in comparison withthe background in order to carry out the separation. By way of example,it is possible to make use of a parallax of the movement betweenforeground and background in order to carry out the separation.Techniques of edge recognition and iterative optimization are carriedout here; cf. ibid.: FIG. 3.

The corresponding disclosure of said article is incorporated bycross-reference herein. In particular, it is possible also to carry outcorresponding techniques for the present separation of the artefact fromthe object. It should be understood here that, in the present case, nomovement of the detector is used to induce the movement of the artefact;rather, the sequence of the reference illumination directions is used toinduce the movement of the artefact. It has nevertheless been recognizedthat it is possible to use corresponding techniques for identifying themovement and for carrying out the artefact reduction.

It is then possible for the method to include, when carrying out theartefact reduction, for each measurement image: combining the respectivemeasurement image with at least one of the assigned reference images onthe basis of the identified movement of the artefact. The combining canmake it possible, for example, to remove the artefact and to reconstructimage regions concealed by the artefact in the measurement image. As aresult, the artefact reduction can be carried out with a high accuracy.

Such techniques which are based on the movement of the artefacttypically have the effect of a particularly high accuracy of theartefact reduction. At the same time, however, the required computingpower may be comparatively high; furthermore, it may be necessary forthe sequence of reference illumination directions to include a verylarge number of reference illumination directions, e.g. more than fiveor more than ten reference illumination directions—, such that capturingthe corresponding reference images may demand a comparatively long timeduration.

Through a suitable choice of the reference illumination directions, itmay be possible to suitably configure the position change of theartefact between the measurement image and a reference image or betweendifferent reference images. In particular, through a suitable choice ofthe reference illumination directions, it may be possible for thisposition change of the artefact to be configured in a characteristicmanner relative to a corresponding position change of the object. Thischaracteristic position change of the artefact can be utilized in theartefact reduction. A description is given below of techniques whichachieve such a suitable choice of the reference illumination directions.

By way of example, it would be possible for the at least one measurementillumination direction which is assigned to a selected measurement imageto form a first average angle with the other measurement illuminationdirections. Said at least one measurement illumination direction of theselected measurement image can form a second average angle with theassigned at least one reference illumination direction, said secondaverage angle being smaller than the first average angle.

It may thus be possible for the different measurement illuminationdirections to form a comparatively large angle with one another; whilethe different reference illumination directions that are assigned to aspecific measurement image form a comparatively small angle with oneanother. By way of example, it would be possible for the differentmeasurement illumination directions which are assigned to differentmeasurement images to form an angle with one another that is greaterthan 20°, preferably >30°, particularly preferably >40°. By way ofexample, it would be possible for the different reference illuminationdirections which are assigned to a specific measurement image to form anangle with one another which is less than 40°, preferably <30°,particularly preferably <20°. For example, it would be possible for thedifferent reference illumination directions which are assigned to aspecific measurement image to form an angle with one another which isless than 15°, preferably <10°, particularly preferably <5°.

What is achieved by means of the comparatively small dimensioning of theangle formed by the at least one reference illumination direction andthe at least one measurement illumination direction is that the objecthas a comparatively small position change between the measurement imageand the reference image; this is the case since the object is typicallyarranged in the focal plane (in a focused manner). At the same time,however, the artefact arranged in a defocused manner can have asignificant position change. In this way, an accurate separation ofartefact and object can be carried out and the artefact reduction can beoperated accurately.

For example, a particularly small dimensioning of the average angleformed by the different reference illumination directions which areassigned to a specific measurement image can be achieved by means ofsuitable driving of the illumination module or of the different lightsources of the illumination module. For example, it would be possiblefor the at least one measurement illumination direction which isassigned to the selected measurement image and the assigned at least onereference illumination direction to correspond to nearest neighbourlight sources of the illumination module. For example, it would thus bepossible for adjacent light sources of the illumination module to beused for generating the measurement image and the assigned referenceimages. Accordingly, it may be possible to use non-closest light sourcesof the illumination module for generating the different measurementimages; this may mean that non-adjacent light sources of theillumination module—that is to say for example light sources of theillumination module between which further light sources are arranged—areused for the different measurement illumination directions.

As a result, it is possible to achieve a comparatively smalldimensioning of the average angle formed by the different referenceillumination directions with one another and with the respective atleast one measurement illumination direction for a specific measurementimage. What can be achieved by means of the comparatively smalldimensions of said angle is that a position change of the objectarranged in a focused manner between the measurement image and the atleast one reference image or between different reference images iscomparatively small. At the same time, however, the position change ofthe artefact may be comparatively large since the artefact arises onaccount of a contaminant which is not arranged in the focal plane of theoptical device.

In general it may be possible for the reference illumination directionsto be at least partly different than the measurement illuminationdirections. This may mean that light sources of the illumination modulethat are controlled for generating the measurement images are differentthan those controlled for generating the reference images. As a result,it may be possible for the information content on which the artefactreduction is based to be particularly large.

In other examples, however, it would also be possible for the referenceillumination directions and the measurement illumination directions tobe chosen to be at least partly identical. In such a case, it may bepossible, for example, that for a specific measurement image one or moreother measurement images are used as the at least one reference imageassigned to the specific measurement image. In this way it may bepossible that the measurement duration can be dimensioned to beparticularly short, such that rapid imaging is possible. The measurementduration can be dimensioned to be short since no or a small number ofdedicated reference images need be captured.

In the various examples described herein, it may be worthwhile, inparticular, for the object—in contrast to the contaminant—to be arrangedin a focal plane of the optical device, that is to say to be arranged ina focused manner. Specifically, what can be achieved in this way is thatthe position change which is observed between the respective measurementimage and the at least one reference image for the artefact ischaracteristic in comparison with the corresponding position change ofthe object. What can be achieved by arranging the object in the focalplane is, for example, that the position change of the object isdimensioned to be comparatively small. Therefore, it would be possible,for example, for the method to further include: driving the sampleholder of the optical device for focusing the object.

In principle, it would be possible for the artefact reduction to becarried out multiple times and iteratively for each measurement image.For example, it would be possible for the artefact reduction to becarried out until a specific convergence criterion is satisfied. Forexample, the convergence criterion can be defined in relation to: numberof iterations; signal-to-noise ratio; and/or duration for carrying outthe artefact reduction. In this way, a particularly accurate artefactreduction can be ensured; while at the same time the measurementduration is not lengthened unnecessarily.

In particular, in the various examples described herein it is possiblefor carrying out the artefact reduction to take place in real time. Inthis way, it may be possible, for example in association with an opticalmicroscope, to image specific real-time processes, for instance thebehavior of cell cultures, of the object in an artefact-reduced manner.

In one example, an optical device has a sample holder, an illuminationmodule, a detector and a computing unit. The sample holder is configuredto fix an object in the beam path of the optical device. Theillumination module has a plurality of light sources. The illuminationmodule is configured to illuminate the object from a plurality ofillumination directions by operating the light sources. The detector isarranged in the beam path of the optical device. The computing unit isconfigured to control the illumination module for illuminating—forexample time-sequentially—an object from a plurality of measurementillumination directions. The computing unit is furthermore configured tocontrol the detector for capturing measurement images of the object,wherein the measurement images are assigned to the measurementillumination directions. The computing unit is furthermore configured,for each measurement image, to carry out an artefact reduction whichreduces an artefact in the respective measurement image on account of acontaminant arranged in defocused fashion. The computing unit isconfigured, after carrying out the artefact reduction for all themeasurement images, to combine the measurement images in order to obtaina result image. The result image can have e.g. a phase contrast.

For such an optical device it is possible to achieve effects that arecomparable with the effects that can be achieved for a method inaccordance with further examples.

By way of example, the optical device can be configured to carry out themethod in accordance with further examples.

In the various examples described herein, different illumination modulescan be used for implementing the angularly-selective illumination or astructured illumination pupil. For example, the illumination modulecould have a carrier, on which the light sources are fitted in a matrixstructure. In this case, the matrix structure could have for exampledifferent unit cells; for example, the matrix structure could have asquare, rectangular or hexagonal unit cell. The number of light sourcesprovided can vary. For example, it would be possible for theillumination module to have more than 10 light sources, preferably morethan 20 light sources, particularly preferably more than 50 lightsources. In this case, different light sources can be used. It would bepossible, for example, for the light sources to be selected from thefollowing group: halogen light sources; light emitting diodes;solid-state light emitting diodes; and organic light emitting diodes.

The properties, features and advantages of this invention describedabove and the way in which they are achieved will become clearer andmore clearly comprehensible in association with the followingdescription of the exemplary embodiments which are explained in greaterdetail in association with the drawings. By way of example, it would bepossible to combine the various examples described above concerning theuse of a filter with techniques of artefact correction by digitalpost-processing.

BRIEF DESCRIPTION OF THE FIGURES

In the drawings:

FIG. 1A schematically illustrates the illumination of a contaminantarranged in a defocused manner from different illumination directionsand an associated position change in corresponding measurement images inaccordance with various embodiments;

FIG. 1B schematically illustrates a result image determined on the basisof a combination of a plurality of measurement images and havingartefacts on account of the contaminant arranged in a defocused manner;

FIG. 2 schematically illustrates the luminous field of an illuminationdirection without a filter;

FIG. 3A schematically illustrates the luminous field of the illuminationdirection from FIG. 2 with a filter in accordance with variousembodiments which is configured to expand the luminous field;

FIG. 3B schematically illustrates the expansion of the luminous field inaccordance with various embodiments in greater detail;

FIG. 4 schematically illustrates an illumination module having aplurality of light sources in accordance with various embodiments,wherein the light sources are implemented as solid-state light emittingdiodes;

FIG. 5 schematically illustrates an illumination module having aplurality of light sources in accordance with various embodiments,wherein the light sources are implemented as organic light emittingdiodes;

FIG. 6 schematically illustrates an illumination module having aplurality of light sources in accordance with various embodiments,wherein the light sources are implemented as halogen light sources;

FIG. 7 schematically illustrates the luminous field which is associatedwith an illumination direction and is expanded by a filter, inaccordance with various embodiments;

FIG. 8 schematically illustrates an optical device in accordance withvarious embodiments;

FIG. 9 schematically illustrates an illumination module having aplurality of light sources in accordance with various embodiments andfurthermore illustrates different measurement illumination directionsand respectively assigned reference illumination directions inaccordance with various embodiments;

FIG. 10 illustrates the measurement illumination directions and thereference illumination directions in accordance with FIG. 9 in greaterdetail;

FIG. 11 schematically illustrates the process of carrying out anartefact reduction on the basis of correction images that are indicativeof the artefact, in accordance with various embodiments;

FIG. 12 schematically illustrates the process of carrying out anartefact reduction on the basis of correction images that are indicativeof the artefact, in accordance with various embodiments;

FIG. 13 schematically illustrates an illumination module having aplurality of light sources in accordance with various embodiments andfurthermore illustrates a sequence of reference illumination directionsfor a measurement illumination direction in accordance with variousembodiments;

FIG. 14 schematically illustrates the movement of the artefact as afunction of the sequence of the reference illumination directions fromFIG. 13;

FIG. 15 is a flowchart of a method in accordance with variousembodiments;

FIG. 16 is a flowchart of a method in accordance with variousembodiments; and

FIG. 17 illustrates result images determined on the basis of an artefactreduction in accordance with various embodiments.

DETAILED DESCRIPTION OF EMBODIMENTS

The present invention is explained in greater detail below on the basisof preferred embodiments with reference to the drawings. In the figures,identical reference signs designate identical or similar elements. Thefigures are schematic representations of different embodiments of theinvention. Elements illustrated in the figures are not necessarilydepicted as true to scale. Rather, the different elements illustrated inthe figures are reproduced in such a way that their function and generalpurpose become comprehensible to the person skilled in the art.Functional units may be implemented as hardware, software or acombination of hardware and software.

Techniques in association with the angularly-selective illumination ofan object are described below. The angularly-selective illumination canbe used e.g. to implement bright-field imaging with a structuredillumination pupil. The techniques described herein can alternatively oradditionally be used in association with phase contrast imaging, whereinthe phase contrast imaging can be carried out by digital post-processingof a plurality of measurement images obtained for different measurementillumination directions. Such techniques are often also referred to asdigital phase contrast imaging with a structured illumination pupil.

Such techniques can be used for example in association with themicroscopy of sample objects. By way of example, such techniques can beused in association with the real-time imaging of sample objects. Inthis case, it may be possible, for example, to implement fluorescenceimaging. The techniques of phase contrast imaging as described hereincan be applied in particular in association with biological sampleobjects having a high phase contrast, but only a limited amplitudecontrast. In general, the techniques described herein can be used e.g.for phase objectives.

The techniques described herein are based on the insight that artefactscan often be amplified during imaging with structured illumination. Inthe case of angularly-selective illumination, discretely arranged lightsources are typically used, e.g. the light sources could be arranged ina matrix arrangement. The discrete distribution of the light sourcesgives rise to gaps in the illumination pupil. Said gaps can result inartefacts in the image that arises in the case of defocused objects.This effect can occur both in traditional bright-field imaging and inphase contrast imaging.

Particularly in phase contrast imaging, such an amplification ofartefacts can take place on account of the combination of the differentmeasurement images in order to obtain a result image in the context ofdigital phase contrast imaging. For example, it has been observed thatthe artefacts present in a corresponding result image can describeextensive patterns that can correlate for example with the structuredillumination used. The pattern can be an image of the illuminationstructure; for example, in the case of a rasterized LED array, arasterized artefact can thus arise for each grain of dust arranged in adefocused manner. On account of the extensive patterns, such artefactscan reduce the usability of the corresponding result image or decreasethe information content of the corresponding result image. As a result,the physico-technical information content of the result image without anartefact reduction in accordance with the techniques described hereinmay be particularly limited.

In the various examples described herein, techniques of artefactreduction can be implemented on the basis of hardware features and/or onthe basis of software features. For example, it is possible for theartefact reduction to be implemented on the basis of a filter, whereinthe filter carries out an expansion of the luminous field that isassigned to a specific illumination direction. By way of example, aplastic diffusing plate can be used as a filter, said diffusing platebeing arranged near the illumination module. As a result, the digitalpost-processing can optionally be carried out by combining themeasurement images to form a result image in accordance with previouslyknown techniques of digital phase contrast imaging. In further examples,it is possible for the artefact reduction to be carried out on the basisof the digital post-processing of measurement images that are combinedto form the result image. The artefact reduction can be carried out forindividual measurement images in order to prevent an amplification ofthe artefacts upon combination to form the result image. This may makeuse for example of the fact that for different reference images whichare associated with a specific measurement image and which are capturedfor different reference illumination directions, a position change ofthe artefact is characteristic in comparison with a position change ofthe object. As a result, it is possible for example, for instance on thebasis of image segmentation techniques, to determine an artefact regionhaving the artefact. It may then be possible to isolate or to remove theartefact in the respective measurement image. However, it is alsopossible, for example, to mark the artefact by taking account of amovement of the artefacts in accordance with a sequence of referenceillumination directions.

FIG. 1A describes aspects with respect to a position change 105 of anartefact 121, 122 depending on the illumination direction 111, 112. Byway of example, an associated measurement image can be captured for eachillumination direction or a measurement image assigned to bothillumination directions 111, 112 is captured.

For example, it would be possible that in the case of illumination alongan illumination direction 111, a detector 101 of a corresponding opticaldevice 100 captures a first measurement image, wherein an artefact 121appears at a first position in the first measurement image (illustratedin FIG. 1A to the right of the optical axis 108). It wouldcorrespondingly be possible that in the case of illumination along theillumination direction 112, the detector 101 captures a secondmeasurement image, wherein an artefact 122 appears at a second positionin the second measurement image. The artefacts 121, 122 are caused bythe same contaminant 120. The contaminant 120 is arranged at a distancefrom a focal plane 109 of an imaging optical unit of the optical device100 (not illustrated in FIG. 1A), i.e. is arranged in a defocusedmanner. FIG. 1A reveals that the position change 105 between theartefacts 121, 122 in the two measurement images results on account ofthis contaminant 120 arranged in a defocused manner.

In principle, such artefacts 121,122 may be observed for contaminants120 in the form of scatterers or absorbers. Typically, artefacts 121,122for contaminants 120 in the form of scatterers appear with brightcontrast in the corresponding image; while artefacts 121,122 forcontaminants 120 in the form of absorbers appear with dark contrast inthe corresponding image.

The position change 105 is given by:

$\begin{matrix}{{{\Delta\; x} = {\Delta\;{z \cdot \frac{\sin( {\alpha + \beta} )}{\cos\mspace{11mu}\alpha\;\cos\mspace{11mu}\beta}}}},} & (1)\end{matrix}$wherein α denotes the angle 111A of the illumination direction 111 withthe optical axis 108, β denotes the angle 112A of the illuminationdirection 112 with the optical axis 108, and Δz denotes the distancebetween the contaminant 120 and the focal plane 109.

Equation 1 can be derived as follows. For the scenario in FIG. 1A itholds true that:Δz=α·cos α=b·cos β,  (2)wherein a denotes a distance between the contaminant 120 and the imaginglocation of the artefact 121 along the illumination direction 111, and bdenotes a distance between the contaminant 120 and the imaging locationof the artefact 122 along the illumination direction 112 (a and b arenot illustrated in FIG. 1A).

By applying the sine law for general triangles, the following isobtained:

$\begin{matrix}{\frac{\Delta\; x}{\sin\;( {\alpha + \beta} )} = {\frac{b}{\sin( {{90{^\circ}} - \alpha} )} = {\frac{b}{\cos\;\alpha}.}}} & (3)\end{matrix}$

Equation (1) is obtained from a combination of equations (2) and (3).Corresponding techniques are also known from DE 10 2014 109 687 A1, thecorresponding disclosure of which is incorporated by cross-referenceherein.

Equation 1 reveals that a larger position change 105 is obtained for agreater defocusing of the contaminant 120, or for larger angles 111A,112A.

FIG. 1B illustrates aspects with regard to a corresponding artefact 123on the basis of actual measurement data. The example in FIG. 1Billustrates in particular two result images obtained by combination ofindividual measurement images captured for different illuminationdirections 111, 112. In the example in FIG. 1B it is evident that theartefact 123 expands or is amplified to form a pattern; this patternimages the structured illumination since the position changes 105between the individual artefacts correlate with the differentillumination directions 111, 112 used (cf. FIG. 1A). The differentillumination directions 111, 112 in turn correlate with the illuminationmodule used, which has a plurality of light sources in a matrixstructure; said matrix structure is imaged in the artefacts 123.

By way of example, corresponding artefacts can also occur in associationwith bright-field imaging. In the case of bright-field imaging,typically a plurality of light sources of a corresponding illuminationmodule are activated per measurement image.

A description is given below of techniques for reducing correspondingartefacts 121-123. With regard to FIGS. 2 and 3A, a hardwareimplementation of a corresponding artefact reduction is illustrated. Inthis case, FIG. 2 illustrates aspects with regard to a luminous field215 of the illumination direction 111 for a reference implementation inwhich no filter is used for expanding the luminous field 215.

It is evident from FIG. 2 that the illumination direction 111 isassociated with a luminous field 215 having a certain extentperpendicular to the corresponding central axis (dashed line in FIG. 2).In the example in FIG. 2, the luminous field 215 of the illuminationdirection 111 is comparatively greatly limited or has a small width.This means that the object arranged in the focal plane 109 (not shown inFIG. 2) is illuminated at a solid angle 111B that is comparativelysmall. The solid angle 111B is centered around the central axis of theillumination direction 111. In various examples, it would be possible,for example, for the size of the solid angle 111B to be limited by anextent of the corresponding light source of the illumination module (notillustrated in FIG. 2) which is used for illumination under theillumination direction 111. For example, lateral dimensions ofsolid-state light emitting diodes may be comparatively limited, suchthat the solid angle 111B also has a comparatively small extent.

FIG. 3A corresponds in principle to FIG. 2, wherein, in the example ofFIG. 3A, a filter 300 is used to expand the luminous field 215 of theillumination direction 111. It is evident from the example in FIG. 3Athat the extent of the luminous field 215 perpendicular to the beam pathof the illumination direction 111 is expanded upon passage through thefilter 300. As a result, the object arranged in the focal plane 109 (notillustrated in FIG. 3A) is illuminated from a comparatively large solidangle 111B.

FIG. 3B illustrates aspects with regard to expanding the luminous field215 of the illumination direction 111 by means of the filter 300. FIG.3B illustrates the amplitude of the luminous field as a function of thelateral position perpendicular to the central axis (dashed line in FIG.3B) of the illumination direction. Moreover, the light propagationdirection is indicated by the arrow. In particular, FIG. 3B illustrateshow the width 217-1 of the luminous field 215 upstream of the filter 300can be expanded in order to obtain the width 217-2 of the luminous field215 downstream of the filter 300 (illustrated on the right-hand side inFIG. 3B). For example, it is possible to define the width 217-1, 217-2of the luminous field 215 in relation to a certain decrease in theamplitude of the luminous field relative to a maximum of the amplitudeof the luminous field 215. Overall, the maximum amplitude of theluminous field may decrease downstream of the filter 300.

For the various techniques of digital phase contrast imaging, acomparatively large solid angle 111B used for illuminating the objectmay bring about no, or no significant, limitation with regard to thequality of the result images obtained in this way. In particular, it maybe possible, owing to the use of greatly different measurementillumination directions, to achieve a pronounced phase contrast of theresult image as a result of the comparatively large dimensioning of thecorresponding angle between the different measurement illuminationdirections. At the same time, however, the use of the comparativelylarge solid angle 111B can have the effect of reducing the individualartefacts 121,122 in the different measurement images in comparison witha smaller solid angle 111B. This may be caused by the defocusedarrangement of the corresponding contaminant: a comparatively largewidth 217-1,217-2 of the luminous field 215 of the correspondingillumination direction 111, 112 brings about a blurring of the contrastfor the individual artefacts 121,122 in the associated measurementimages.

FIG. 4 illustrates aspects with regard to an illumination module 180which can be used for the angularly-selective illumination fromdifferent illumination directions. The illumination module 180 has acarrier 181, for example composed of metal or plastic. A plurality oflight sources 182 are arranged on the carrier. In the example in FIG. 4,the different light sources 182 are arranged in a matrix structurehaving a hexagonal unit cell on the carrier 181. Other matrix structuresare also possible, for example having a square unit cell. For example,it would be possible for the illumination module 180 to be arrangedcentrally in the region of the optical axis of the optical device 100.

Operating the different light sources 182 makes it possible to implementthe illumination of the object from different illumination directions.The greater a distance between the different light sources 182,typically the greater an angle formed by the different illuminationdirections with one another. In this case, one or more light sources 182can be activated per measurement image.

A wide variety of types of light sources 182 can be used. In the examplein FIG. 4, solid-state light emitting diodes, for instance, are used aslight sources 182. The solid-state light emitting diodes have acomparatively limited lateral extent.

FIG. 5 illustrates aspects in relation to an illumination module 180. Inthe example in FIG. 5, the light sources 182 are configured as organiclight emitting diodes. These organic light emitting diodes 182 have asignificant lateral extent, for example in comparison with thesolid-state light emitting diodes which implement the light sources 182in the example in FIG. 4. What can be achieved by the use of laterallycomparatively extensive light sources 182, as in the example in FIG. 5,is that the luminous field associated with the different correspondingillumination directions already has a comparatively large width. It maythen be possible that the filter 300, for example, must perform arelatively small expansion in order to achieve an efficient artefactreduction. As a result, it is possible to limit a reduction of themaximum amplitude of the luminous field by the filter 300. This mayincrease a signal-to-noise ratio.

FIG. 6 illustrates aspects in relation to an illumination module 180. Inthe example in FIG. 6, the light sources 182 are embodied as halogenlight sources. The halogen light sources also have a comparatively largelateral extent, for example in comparison with the solid-state lightemitting diodes which implement the light sources 182 in the example inFIG. 4.

FIG. 7 illustrates aspects with regard to the filter 300. In the examplein FIG. 7, the filter 300 is arranged in the beam path of the opticaldevice 100 between a sample holder 102 and the illumination module 180.In particular, in the example in FIG. 7, the filter 300 is arrangedbetween an imaging optical unit 185, e.g. an objective or a collimatoroptical unit—of the optical device 100 and the illumination module 180.The filter is configured to expand the assigned luminous field 215 forthe different illumination directions—which can be implemented byoperating different light sources 182 of the illumination module 180. Byway of example, the filter 300 could have a diffusing plate composed ofplastic.

In the example in FIG. 7, the filter 300 is arranged in close proximityto the carrier 181 of the illumination module 180. In particular, in theexample in FIG. 7, the carrier 181 is rigidly coupled to the filter 300.Such techniques make it possible to carry out a particularly efficientexpansion of the luminous field 215 of the illumination direction 111(in FIG. 7, the non-expanded luminous field 215 is illustrated by adashed line and the expanded luminous field 215 is illustrated by asolid line).

The optical device 100 can furthermore have a detector (not illustratedin FIG. 7). The detector can be arranged for example usingreflected-light geometry, i.e. in FIG. 7 on the left-hand side of thesample holder 102 in the region of the reflected beam path. However, itwould also be possible for the detector to be arranged usingtransmitted-light geometry, that is to say in FIG. 7 on the right-handside of the sample holder 102 in the region of the transmitted beampath.

FIG. 8 illustrates aspects with regard to the optical device 100. Forexample, the optical device 101 could implement a microscope. Forexample, the optical device 100 could be configured for fluorescenceimaging. For example, the optical device could implement a laserscanning microscope.

The optical device 100 has the illumination module 180, the sampleholder 102 and a detector 101. As described above, the detector can bearranged for example using reflected-light geometry or transmitted-lightgeometry with respect to the illumination module 180 and the sampleholder 102. For example, the detector can be a CCD detector or a CMOSdetector.

The optical device 100 also has a computing unit 103, for example aprocessor and/or a computer and/or an ASIC. The computing unit 103 isconfigured to control the illumination module 180 and to control thedetector 101. Optionally, the computing unit 103 could also beconfigured to control the sample holder 102 for focusing the object; amanual focusing of the object by way of adjusting the sample holder 102by hand would also be conceivable.

By means of the computing unit 103 it is possible to carry out a digitalpost-processing of images that are captured by the detector 101. Forexample, the computing unit could be configured to control theillumination module 180 for illuminating the object from a plurality ofmeasurement illumination directions. The computing unit 103 can also beconfigured to control the detector for capturing measurement images ofthe object, wherein the measurement images are assigned to themeasurement illumination directions.

Different assignments between the measurement images and the measurementillumination directions are possible, that is to say that a 1:nassignment with n>=1 can be implemented. In this case, n can vary or beidentical for different measurement images. In this case, it is possibleto carry out a separation of the measurement illumination directions forthe different measurement images e.g. in the time domain, color space orpolarization space. That means that it would be possible, for example,to process time-sequentially the measurement illumination directionsassigned to the different measurement images. Alternatively oradditionally, however, it would also be possible to capture measurementimages at least partly in a time-parallel manner; in this case, it ispossible to differentiate between the different measurement illuminationdirections e.g. by way of the spectral range, i.e. the color, of thelight and/or the polarization. Corresponding filters can be provided.

The computing unit could then furthermore be configured to combine themeasurement images in order to obtain a result image. What can beachieved by combining the measurement images associated with differentillumination directions is that the result image has a phase contrast.By means of suitable combination, a wide variety of conventional phasecontrast techniques can be simulated or emulated, for example phasecontrast according to Waller, DPC phase contrast, Zernike phasecontrast, etc. Bright-field imaging can also be implemented.

While a hardware-based artefact reduction can be carried out—in realtime—with the use of the filter 300, in various examples it mayalternatively or additionally be possible for the computing unit 103 tobe configured for a software-based artefact reduction. Thesoftware-based artefact reduction can also be carried out in real timein various examples. For example, it would be possible to carry out, foreach measurement image, an artefact reduction that reduces acorresponding artefact 121, 122 in the respective measurement image onaccount of a contaminant 120 arranged in a defocused manner. Then, aftercarrying out the artefact reduction for all the measurement images, itis possible for the measurement images to be combined in order to obtaina result image having a phase contrast.

In order to carry out a particularly accurate software-based artefactreduction, it may be possible to take into account, in addition to themeasurement images—on the basis of which the result image isdetermined—, reference images associated with reference illuminationdirections. The information basis for the artefact reduction can beextended as a result.

FIG. 9 illustrates aspects with regard to reference illuminationdirections 411-413, 421-423, 431-433, 441-443. The example in FIG. 9illustrates how the illumination module 180, for different measurementimages associated with different measurement illumination directions111-114, can be controlled for illuminating the object from respectivelythree assigned reference illumination directions 411-413, 421-423,431-433, 441-443. For each reference illumination direction 411-413,421-423, 431-433, 441-443, the detector 101 can then be driven tocapture an associated reference image of the object. In this case, e.g.the measurement illumination direction 111 is assigned to the referenceillumination directions 411-413. In this case, e.g. the measurementillumination direction 112 is assigned to the reference illuminationdirections 421-423. In this case, e.g. the measurement illuminationdirection 113 is assigned to the reference illumination directions431-433. In this case, e.g. the measurement illumination direction 114is assigned to the reference illumination directions 441-443.

It is evident from FIG. 9 that the reference illumination directions411-413, 421-423, 431-433, 441-443 are grouped in each case around thecorresponding measurement illumination direction 111-114, whereinhowever, the reference illumination directions 411-413, 421-423,431-433, 441-443 are different than the measurement illuminationdirections 111-114. In particular, the measurement illuminationdirections 111-114 and the assigned reference illumination directions411-413, 421-423, 431-433, 441-443 correspond to light sources 182 ofthe illumination module 180 that are closest to one another. What can beachieved as a result is that an average angle formed by the differentreference illumination directions 411-413, 421-423, 431-433, 441-443assigned to a measurement illumination direction 111-114 with oneanother is smaller than an average angle formed by the differentmeasurement illumination directions 111-114 with one another.

FIG. 10 illustrates aspects with regard to angles 451, 452 betweenreference illumination directions 411,412 and measurement illuminationdirections 111-114. In particular, FIG. 10 illustrates a perspectiveview of the scenario from FIG. 9, wherein FIG. 10 does not illustrateall of the reference illumination directions, for reasons of clarity.

FIG. 10 illustrates an angle 452 formed by the measurement illuminationdirection 111 with the reference illumination direction 411. It isevident from FIG. 10 that said angle 452 is significantly smaller thanan angle 451 formed by the measurement illumination direction 111 withthe measurement illumination direction 112. The same correspondinglyalso applies to the average angle between the measurement illuminationdirection and the reference illumination directions 411-413. This is thecase since non-adjacent light sources 182 of the illumination module 180are used for the different measurement illumination directions 111-114(cf. FIG. 9); while light sources 182 of the illumination module 180that are closest to the respective light source associated with thecorresponding measurement illumination direction 111 are used for thereference illumination directions 411-412.

FIG. 11 illustrates aspects with regard to a digital artefact reduction.In the example in FIG. 11, the artefact reduction is based on the use ofa reference illumination direction 411 to which a correspondingreference image 551 is assigned. FIG. 11 also illustrates the associatedmeasurement image 501. The different images 501,551 image the object 125and have artefacts 121. Moreover, the images 501, 551 have a background(illustrated in a diagonally striped manner in FIG. 11). The aim of theartefact reduction is to suppress the artefacts 121, but to preserve asfar as possible the object 125 and the background.

It is evident from a comparison of the measurement image 501 with theassociated reference image 551 that the artefacts 121 have significantlychanged their position, while the object 125 remains substantially fixedin place. This is the case since the associated contaminant 120 (notillustrated in FIG. 11) is arranged in a defocused manner outside thefocal plane 109 of the optical device 100 and, as a result, theinfluence on the position change as a result of the angle between thereference illumination direction 411 and the measurement illuminationdirection 111 for the imaging of the contaminant 120 as the artefact 121is particularly great (cf. equation 1). Since the object 125 is arrangedin the focal plane 109, it has no, or no significant, position changebetween the measurement image 501 and the reference image 551 (cf.equation 1); it should be understood here that for an object 125extended in the depth direction (i.e. parallel to the beam path) theremay also be a certain position change between the measurement image 111and the reference image 411, in particular for regions of the object 125that are arranged in a defocused manner. Therefore, it may beworthwhile, in principle, to dimension the angle 452 between thereference illumination direction 411 and the measurement illuminationdirection 111 such that it is comparatively small, in order to result inthe smallest possible dimensioning of said position change with respectto the object 125.

In the example in FIG. 11, combining the measurement image 501 with theassigned reference image 551 in order to obtain a correction image 563is carried out. In the example in FIG. 11, the combining is carried outby subtracting the reference image 551 from the measurement image 501.The correction image 563 in the example in FIG. 11 then has a negativecontrast (shown black in FIG. 11) and a positive contrast (shown whitein FIG. 11) for the artefacts 121, wherein the negative contrast isassociated with the position of the artefacts in the measurement image501 and the positive contrast is associated with the position of theartefacts 121 in the reference image 551. The object 125 is stillpresent in outline form in the correction image 563 since, as describedabove, there may be a small position change of the object 125 betweenthe measurement image 501 and the reference image 551. The samecorrespondingly applies to the background. However, the intensity of theobject 125 and of the background is greatly reduced.

An image segmentation is then applied to the correction image 563; inthis case, the image segmentation is based on an intensity thresholdvalue. On the basis of the image segmentation it is possible to obtainan isolated artefact region in the correction image 564, said artefactregion including the artefact 121. The artefact 121 can then be removedfrom the measurement image 501 by once again subtracting the artefactregion of the correction image 564 from the measurement image 501 inorder to obtain a corrected measurement image 502.

In the example in FIG. 11, the contaminants 120 are absorbers;therefore, the artefacts 121 appear with negative contrast in themeasurement image 501 and the reference image 551. In other examples itis also possible for the contaminants to be scatterers, such that theartefacts appear with positive contrast in the corresponding measurementimages and reference images.

In some examples, it may also be possible for the contaminants 120 tohave both absorbers and scatterers. One such example is illustrated withreference to FIG. 12. FIG. 12 illustrates aspects in relation to digitalartefact reduction.

The scenario in FIG. 12 corresponds, in principle, to the scenario inFIG. 11, but two reference images 551, 552 are used instead of a singlereference image, said reference images being captured for respectivelydifferent reference illumination directions 411,412. By using a greaternumber of reference illumination directions 411, 412 or a greater numberof reference images 551, 552, it may be possible to carry out aparticularly accurate artefact reduction even in the presence ofartefacts 121 corresponding to scatterers and absorbers.

In detail, a correction image 563-1 is obtained by difference formationbetween the measurement image 501 and the reference image 551. Acorrection image 563-2 is obtained by difference formation between themeasurement image 501 and the reference image 552. The correction images564-1 and 565-1 are obtained respectively by image segmentation of thecorrection image 563-1 relating to a positive and negative intensitythreshold value. The correction images 564-2 and 565-2 are obtainedrespectively by image segmentation of the correction image 563-2relating to a positive and negative intensity threshold value. Thecorrection images 564-1, 564-2, 565-1, 565-2 therefore define isolatedartefact regions corresponding to the different artefacts 121. In thiscase, the different artefact regions of the correction images 564-1 and565-1 are subsequently corrected on the basis of the artefact regions ofthe correction images 564-2 and 565-2 (or the other way around) in orderto obtain the correction images 566 and 567. In detail, the correctionimage 566 is generated by applying a further image segmentation on thebasis of a positive intensity threshold value with respect to thecorrection images 565-1 and 565-2. The correction image 567 is generatedby applying a further image segmentation on the basis of a negativeintensity threshold value with respect to the correction images 564-1and 564-2.

As a result, it is possible to determine the artefacts 121 in themeasurement image 501 which respectively correspond to absorbers(correction image 566) and scatterers (correction image 567). This canbe utilized in order to generate a corrected measurement image 502 bycombining the measurement image 501 with the correction images 566 and567.

FIG. 13 illustrates aspects with regard to a sequence 499 of referenceillumination directions 411-418. The reference illumination directions411-418 are assigned to the measurement illumination direction 111 (inFIG. 13, the reference illumination directions for the measurementillumination directions 112-114 are not illustrated, for reasons ofclarity). It is evident from FIG. 13 that the sequence 499 is definedwith respect to a spatial succession of the reference illuminationdirections 411-418. Adjacent elements of the sequence 499 thereforecorrespond to adjacent reference illumination directions 411-418. Inthis case, it is unnecessary in principle for a temporal order withwhich the sequence 499 is processed and corresponding reference imagesare captured to correspond to said spatial succession of the sequence499.

FIG. 14 illustrates the different reference images 551-558 correspondingto the reference illumination directions 411-418 of the sequence 499. Itis evident from FIG. 14 that on account of the spatial succession thereis a systematic movement of the artefact 121 in the different referenceimages 551-558. It is possible to identify the movement of the artefact121 as a function of the respective sequence 499 in the reference images551-558. It may then be possible for the artefact reduction to becarried out on the basis of the identified movement of the artefact. Forexample, it would be possible that, given identified movement of theartefact 121, the artefact 121 can be computationally extracted from themeasurement image 501. To that end, it may be possible for themeasurement image 501 to be combined with at least one of the assignedreference images 551-558 on the basis of the identified movement of theartefact 121.

In this case, various techniques can be used for identifying themovement of the artefact 121 in the sequence 499. Techniques of imagesegmentation and/or edge recognition can be used, for example.Optimization techniques can also be used. Corresponding techniques foridentifying the movement are known for example from the article by XUET. et al. cited above.

FIG. 15 is a flowchart of one exemplary method. In this case, steps 1001and 1002 involve respectively driving the illumination module 180 andthe detector 101 in order to capture a respective associated measurementimage 501 for different measurement illumination directions 111-114. Inthis case, it is possible to implement more than one illuminationdirection per measurement image 501: to that end, e.g. in 1001 theillumination module 180 can be controlled in such a way that more thanone light source 182 is activated.

1003 involves checking whether a further measurement image 501 isrequired; if this is the case, then steps 1001 and 1002 are carried outagain. For example, in 1003 it is possible to take account of whetherphase contrast imaging is intended to be carried out—and if so, whattype of phase contrast imaging is intended to be employed. By way ofexample, a different number of measurement images 501 may be requireddepending on the type of phase contrast imaging.

If it is ascertained in step 1003 that no further measurement image 501is required, then step 1004 involves carrying out the artefact reductionfor each measurement image 501 captured previously in the differentiterations of step 1002, in order to obtain a corrected measurementimage 502 in each case.

The artefact-reduced measurement images 502 are then combined with oneanother in step 1005 in order to obtain a result image. The result imagemay optionally have a phase contrast.

In various examples, it would be possible for the artefact reduction instep 1004 to be carried out multiply, i.e. in a plurality of iterations.For example, it would be possible for an artefact-reduced measurementimage 502 to be generated in each case in a plurality of iterations. Forexample, the operations described above with reference to FIGS. 11 and12 could be carried out multiple times and iteratively in order toachieve an improved artefact reduction in each case.

Step 1004 could e.g. also be carried out before step 1003, i.e. in eachcase for the measurement image captured previously.

A wide variety of techniques can be used for carrying out the artefactreduction in step 1004. By way of example, it may be possible todetermine, on the basis of one or more reference images 551, 552, acorrection image that is indicative of the artefact 121 in theassociated measurement image 501; to that end, an image segmentation canbe carried out, for example, as explained above with reference to FIGS.11 and 12. Alternatively or additionally, it would also be possible tocapture a sequence 499 of reference images 551-558 and to identify amovement of the artefact 121 in the sequence 499. The artefact reductioncan then be carried out on the basis of the identified movement. Suchtechniques at all events involve capturing one or more reference images551-558.

FIG. 16 is a flowchart of a method in accordance with various examples.FIG. 16 illustrates aspects with regard to capturing one or morereference images 551-558.

In this case, firstly, in step 1011, one or more reference illuminationdirections 411-418 are selected for a current measurement illuminationdirection 111 or for a current measurement image 501.

Steps 1012 and 1013 then involve driving the illumination module 180 andthe detector 101 in a time-synchronized manner in order to capture anassociated reference image 551-558 in each case for a current referenceillumination direction 411-418. Step 1014 involves checking whether afurther reference image 551-558 need be captured; if this is the case,steps 1012 and 1013 are carried out again.

In some examples, a single reference illumination direction 411-418 canbe implemented for each reference image 551-558. In other examples,however, it would also be possible for more than a single referenceillumination direction 411-418 to be assigned to at least some referenceimages 551-558. To that end, e.g. in 1012, the illumination module 180can be controlled in such a way that more than one light source 182 isactivated.

Step 1015 involves checking whether one or more reference images shouldbe captured for a further measurement image 501. If this is the case,steps 1011-1014 are carried out again.

Steps 1011-1015 for capturing the reference images 551-558 can becarried out for example before or after capturing the differentmeasurement images 502, i.e. before or after carrying out steps 1001 and1002. It would also be possible for capturing the reference images551-558 to be carried out in a manner temporally overlapping thecapturing of the measurement images 502. This may be worthwhileparticularly in the case of temporally variable sample objects, in orderto avoid movement artefacts.

FIG. 17 illustrates aspects with regard to result images 601, obtainedon the basis of the combination of different measurement images whichwere previously corrected on the basis of one or more iterations of theartefact reduction as described above with reference to FIG. 12.Uncorrected result images 601A are also illustrated for comparison. Itis evident from FIG. 17 that the quality of the artefact reductionincreases for a larger number of iterations. However, a significantsuppression of the artefact 123 can be achieved even with one iterationof the artefact reduction.

To summarize, a description has been given above of techniques forcarrying out artefact reduction during angularly-selective illuminationwith a plurality of discretely arranged light sources. The techniquesare based on the insight that contaminants arranged in a defocusedmanner during the angularly-selective illumination are amplified to formextensive artefacts having a pattern corresponding to the structuredillumination pupil.

Although the invention has been more specifically illustrated anddescribed in detail by means of the preferred exemplary embodiments,nevertheless the invention is not restricted by the examples disclosedand other variations can be derived therefrom by the person skilled inthe art, without departing from the scope of protection of theinvention.

By way of example, a description has been given above of variousexamples with regard to phase contrast imaging. However, it is possiblefor the techniques described herein e.g. also to be applied toconventional bright-field imaging in which a structured illumination isused. In this case, during bright-field imaging it may be worthwhile toactivate as many as possible or all of the light sources of thecorresponding illumination module in order in this way to achieve auniform illumination of the object from different spatial directions.

Furthermore, a description has been given above of various examples inwhich the different measurement images are assigned in each case to asingle measurement illumination direction. In other examples, however,it would also be possible for the different measurement images to beassigned to more than a single measurement illumination direction, e.g.two, three or more measurement illumination directions. In such cases,too, the techniques for artefact correction as described herein can beapplied.

While the invention has been illustrated and described in connectionwith currently preferred embodiments shown and described in detail, itis not intended to be limited to the details shown since variousmodifications and structural changes may be made without departing inany way from the spirit of the present invention. The embodiments werechosen and described in order to best explain the principles of theinvention and practical application to thereby enable a person skilledin the art to best utilize the invention and various embodiments withvarious modifications as are suited to the particular use contemplated.

LIST OF REFERENCE SIGNS

-   111-114 Illumination direction-   100 Optical device-   101 Detector-   102 Sample holder-   103 Computing unit-   105 Position change-   108 Optical axis-   109 Focal plane-   111A Angle-   111B Angle-   120 Contaminant-   121 Artefact-   122 Artefact-   123 Artefact-   125 Object-   180 Illumination module-   181 Carrier-   182 Light source-   185 Imaging optical unit-   215 Luminous field-   217-2 Width of the luminous field-   217-1 Width of the luminous field-   300 Filter-   451 Angle-   452 Angle-   501 Measurement image-   502 Corrected measurement image-   566 Correction image-   567 Correction image-   601 Result image-   601A Result image-   411-418 Illumination direction-   421-423 Illumination direction-   431-433 Illumination direction-   441-443 Illumination direction-   551-558 Reference image-   565-1, 565-2 Correction image-   563, 563-1, 563-2 Correction image-   564, 564-1, 564-2 Correction image-   1001-1005 Step-   1011-1015 Step

What is claimed is:
 1. A method, comprising: controlling an illuminationmodule of an optical device having a plurality of light sources forilluminating an object from a plurality of measurement illuminationdirections; controlling a detector of the optical device for capturingmeasurement images of the object, wherein the measurement images areassigned to the measurement illumination directions; carrying out, foreach measurement image, an artefact reduction which reduces an artefactin the respective measurement image on account of a contaminant arrangedin defocused fashion; after carrying out the artefact reductions for allthe measurement images, combining the measurement images in order toobtain a result image; driving, for each measurement image, theillumination module for illuminating the object from at least oneassigned reference illumination direction; driving, for each referenceillumination direction, the detector for capturing a reference image ofthe object; when carrying out the artefact reduction, for eachmeasurement image, combining the respective measurement image with theat least one assigned reference image in order to obtain at least onecorrection image which is indicative of the artefact; wherein thecontaminant comprises scatterers and absorbers, wherein the illuminationmodule is driven for illuminating the object from at least two assignedreference illumination directions for each measurement image; andwherein the method further comprises: when carrying out the artefactreduction, for each correction image, correcting the respective artefactregion on the basis of the artefact region of a further correctionimage.
 2. The method as claimed in claim 1, further comprising: whencarrying out the artefact reduction, for each correction image, applyingan image segmentation on the basis of an intensity threshold value inorder to obtain an isolated artefact region in the correction image,said artefact region including the artefact; and when carrying out theartefact reduction, for each measurement image, removing the artefact onthe basis of the artefact region.
 3. The method as claimed in claim 1,further comprising: driving, for each measurement image, theillumination module for illuminating the object from an assignedsequence of reference illumination directions; driving, for eachreference illumination direction, the detector for capturing a referenceimage of the object; and when carrying out the artefact reduction, foreach measurement image, identifying a movement of the artefact as afunction of the respective sequence of the reference illuminationdirections in the assigned reference images and wherein the respectiveartefact reduction is based on the respectively identified movement ofthe artefact.
 4. The method as claimed in claim 3, further comprising:when carrying out the artefact reduction, for each measurement image,combining the respective measurement image with at least one of theassigned reference images on the basis of the identified movement of theartefact.
 5. The method as claimed in claim 1, wherein the at least onemeasurement illumination direction which is assigned to a selectedmeasurement image forms a first average angle with the other measurementillumination directions, and wherein the at least one measurementillumination direction which is assigned to the selected measurementimage forms a second average angle with the assigned at least onereference illumination direction, said second average angle beingsmaller than the first average angle.
 6. The method as claimed in claim1, wherein the at least one measurement illumination direction which isassigned to the selected measurement image and the assigned at least onereference illumination direction corresponds to nearest neighbor lightsources of the illumination module.
 7. The method as claimed in claim 1,wherein the reference illumination directions are at least partlydifferent from the measurement illumination directions.
 8. The method asclaimed in claim 1, further comprising: controlling a sample holder ofthe optical device, said sample holder being configured to fix theobject in the beam path of the optical device, for focusing the object.9. The method as claimed in claim 1, wherein the artefact reduction iscarried out multiple times and iteratively for each measurement image.10. The method as claimed in claim 1, wherein carrying out the artefactreduction occurs in real time.
 11. An optical device, comprising: asample holder configured to fix an object in the beam path of theoptical device; an illumination module having a plurality of lightsources and configured to illuminate the object from a plurality ofillumination directions by operating the light sources; a detectorarranged in the beam path of the optical device; and a computing unitconfigured to control the illumination module for illuminating an objectfrom a plurality of measurement illumination directions; wherein thecomputing unit is configured to control the detector for capturingmeasurement images of the object, wherein the measurement images areassigned to the measurement illumination directions, wherein thecomputing unit is configured, for each measurement image, to carry outan artefact reduction which reduces an artefact in the respectivemeasurement image on account of a contaminant arranged in defocusedfashion, wherein the computing unit is configured, after carrying outthe artefact reduction for all the measurement images, to combine themeasurement images in order to obtain a result image; driving, for eachmeasurement image, the illumination module for illuminating the objectfrom at least one assigned reference illumination direction; driving,for each reference illumination direction, the detector for capturing areference image of the object; when carrying out the artefact reduction,for each measurement image, combining the respective measurement imagewith the at least one assigned reference image in order to obtain atleast one correction image which is indicative of the artefact; whereinthe contaminant comprises scatterers and absorbers, wherein theillumination module is driven for illuminating the object from at leasttwo assigned reference illumination directions for each measurementimage; and wherein the method further comprises: when carrying out theartefact reduction, for each correction image, correcting the respectiveartefact region on the basis of the artefact region of a furthercorrection image.
 12. The optical device as claimed in claim 11, whereinthe optical device is configured to carry out the method as claimed inclaim 1.